Apoptosis is absolutely necessary for human development and survival, with millions of cells committing suicide daily as a way to prevent uncontrolled growth. Defects in apoptosis, together with amplified growth signals, often lead to cancer. Targeting apoptosis defects in cancer has a tremendous potential.
The first human apoptotic protein identified was BCL-2, as inhibitor of apoptosis, in 1984. The role of caspases-proteases that act as the cell's direct executioners by cleaving other cellular proteins was revealed in humans beginning in 1993. In cell ready to die, pro-apoptotic BCL-2 family members, like BAX, disrupt mitochondria, causing the release of other proteins that lead to caspase release and cell death. Activation of this so-called “intrinsic” apoptotic pathway is the goal of many of the new cancer drugs.
A second, “extrinsic”, cell death pathway is also an important target, and the first so-called death receptor, DR4, was discovered around 1996.
Most of the current cancer therapies, including chemotherapeutic agents, radiation, and immunotherapy, work by indirectly inducing apoptosis in cancer cells. The inability of cancer cells to execute an apoptotic program due to defects in the normal apoptotic machinery is thus often associated with an increase in resistance to chemotherapy, radiation or immunotherapy-induced apoptosis.
In this regard, targeting crucial negative regulators that play a central role in directly inhibiting apoptosis in cancer cells represents a highly promising therapeutic strategy for new anticancer drug design.
Two classes of central negative regulators of apoptosis have been identified. The first class is the Bcl-2 family of proteins (Adams et al., Science 281:1322, 1998; Reed, Adv. Pharmacol. 41:501, 1997; Reed et al., J. Cell. Biochem. 60:23, 1996). Currently, Bcl-2 antisense therapy is in several Phase III clinical trias for the treatment of solid and not solid tumors.
The second class of central negative regulators of apoptosis is the inhibitor of apoptosis proteins (IAPs) (Deveraux et al., Genes Dev. 13:239, 1999; Salvesen et al., Nat. Rev. Mol. Cell. Biol. 3:401, 2002). IAPs potently suppress apoptosis induced by a large variety of apoptotic stimuli, including chemotherapeutic agents, radiation, and immunotherapy in cancer cells.
X-linked IAP (XIAP) is the most potent inhibitor in suppressing apoptosis among all of the IAP members (Holcik et al., Apoptosis 6:253, 2001; LaClasse et al., Oncogene 17:3247, 1998; Takahashi et al., J. Biol. Chem. 273:7787, 1998; Deveraux et al., Nature 388:300, 1997; Sun et al., Nature 401:818, 1999; Deveraux et al., EMBO J. 18:5242, 1999; Asselin et al., Cancer Res. 61:1862, 2001). XIAP plays a key role in the negative regulation of apoptosis in both the death receptor-mediated and the mitochondria-mediated pathways. XIAP functions as a potent endogenous apoptosis inhibitor by directly binding and potently inhibiting three members of the caspase family enzymes, caspase-3, -7, and -9 (Takahashi et al., J. Biol. Chem. 273:7787, 1998; Deveraux et al., Nature 388:300, 1997; Sun et al., Nature 401:818, 1999; Deveraux et al., EMBO J. 18:5242; Asselin et al., Cancer Res. 61:1862, 2001; Riedl et al., Cell 104:791, 2001; Chai et al., Cell 104:769, 2001; Huang et al., Cell 104:781, 2001). XIAP contains three baculovirus inhibitor of apoptosis repeat (BIR) domains. The third BIR domain (BIR3) selectively targets caspase-9, the initiator caspase in the mitochondrial pathway, whereas the linker region between BIR1 and BIR2 inhibits both caspase-3 and caspase-7 (Salvesen et al., Nat. Rev. Mol. Cell. Biol. 3:401, 2002). While binding to XIAP prevents the activation of all three caspases, it is apparent that the interaction with caspase-9 is the most critical for its inhibition of apoptosis (Ekert et al., J. Cell Biol. 152:483, 2001; Srinivasula et al., Nature 410:112, 2001). Because XIAP blocks apoptosis at the downstream effector phase, a point where multiple signalling pathways converge, strategies targeting XIAP may prove to be especially effective to overcome resistance of cancer cells to apoptosis (Fulda et al., Nature Med. 8:808, 2002; Arnt et al., J. Biol. Chem. 277:44236.2002).
There are evidence to indicate that XIAP is widely overexpressed in many types of cancer and may ply an important role in the resistance of cancer cells to a variety of current therapeutic agents (Holcik et al., Apoptosis 6:253, 2001; LaClasse et al., Oncogene 17:3247, 1998).
Recently, Smac/DIABLO (second mitochondria-derived activator of caspases) was identified as a protein released from mitochondria into the cytosol in response to apoptotic stimuli (Budihardjo et al., Annu. Rev. Cell Dev. Biol. 15:269, 1999; Due t al., Cell 102:33, 2000). Smac is synthesized with an N-terminal mitochondrial targeting sequence that is proteolytically removed durino maturation to the mature polipeptide. Smac was shown to directly interact with XIAP and other IAPs and to disrupt their binding to caspases and facilitate caspases activation. Smac is a potent endogenous inhibitor of XIAP.
Smac/DIABLO interacts with both the BIR2 and BIR3 domains of XIAP (Chai. J. et al. Nature 406:855, 2000). The crystal structure of Smac/DIABLO reveals that it forms a homodimer through a large, hydrophobic interface, and that homodimerization is essential for its binding to the BIR2, but not BIR3, domain of XIAP (Chai. J. et al. Nature 406:855, 2000). The four amino-terminal residues of Smac/DIABLO (Ala-Val-Pro-Ile) make specific contact with a surface groove of the BIR2 and BIR3 domains, but not with the BIR1 domain, of XIAP (Wu, G. et at Nature 408:1008, 2000; Liu, Z. et al. Nature 408:1004, 2000). Significantly, the conserved tetrapeptide motif has remarkable homology to the IAP-interacting motif found in the p12 amino-terminal sequence of caspase-9 (Ala-Thr-Pro-Phe) and the Drosophila proteins Hid (Ala-Val-Pro-Phe), Reaper (Ala-Val-Ala-Phe) and Grim (Ala-Ile-Ala-Tyr).
The Kd value of Smac peptide AVPI to XIAP (Kd=0.4 μM) is essentially the same as the mature Smac protein (Kd=0.42 μM).